Technical Abstract:
The identification of proteins by tandem mass spectrometry (MS/MS) is based on the best fit of spectral data to databases of protein sequences. Although current databases contain sequences for many wheat gluten proteins, these represent only a portion of the sequence heterogeneity present in the gluten protein families. To distinguish individual gliadins from the US wheat Butte 86, ESTs for alpha and gamma gliadins from Butte 86 were identified in public databases and assembled into contigs. Consensus sequences from 12 of
19 alpha gliadin contigs and nine of 11 gamma gliadin contigs encoded full-length proteins. Differences in epitopes related to celiac disease were observed among the proteins and two new types of alpha gliadins ending in GFFGTN and GIMSTN were discovered. In addition, the analysis revealed one alpha gliadin and four gamma gliadins containing an odd number of cysteine residues that would enable the proteins to be incorporated into the glutenin polymer. By including the Butte 86 alpha and gamma gliadin sequences in a protein database used for analysis of MS/MS data obtained from trypsin, chymotrypsin or thermolysin digestion of wheat flour proteins, 12 closely related alpha gliadin and 5 gamma gliadin proteins were identified in Butte
86 flour with high levels of confidence. This approach made it possible to distinguish closely-related gliadins in Butte 86 that differ in epitopes important in celiac disease as well as gliadins that include an extra cysteine residue and could function as chain terminators of the glutenin polymer.